The Src homology 2 domain-containing gene 1A (SH2D1A) encodes an adaptor molecule known as SAP that is defective in patients with X-linked lymphoproliferative disease (XLP), a disorder associated with abnormal antiviral and antitumor immunity. SAP is expressed in T cells and Natural Killer (NK) cells, where it regulates Thl and Th2 cytokine production, as well as cytotoxic function. To determine whether SAP controls the activity of NKT cells, a lymphocyte subset sharing features with T and NK cells, we examined SAP -/- mice and human XLP patients for the presence of this lineage. Remarkably, NKT cells were dramatically reduced or absent, suggesting that, in addition to its essential role in mature T and NK cells, SAP is required for NKT cell development. Consistent with the lack of NKT cells, SAP -/- mice failed to upregulate cytokines in response to a-galactosyl ceramide, a glycolipid antigen that specifically activates these cells. Based on these new findings, we hypothesize that SAP is a critical signaling molecule that is required for coordinating the biochemical pathways controlling NKT cell ontogeny. We also propose that the severe reduction in NKT cells, when combined with abnormally functioning T and NK cells, contributes to the pathogenesis of XLP. In this proposal, we aim to rigorously dissect the role of SAP during NKT cell development and mature NKT cell activation. To firmly establish whether SAP is required during human NKT cell differentiation, in Aim 1 we will use cell-based and molecular assays to evaluate NKT cell number in a larger spectrum of patients with XLP and related immunological defects. In Aim 2, we will study whether SAP is required for mature NKT cell function. After reconstituting NKT cell development in SAP -/- mice, WT and SAP -/-cells will be investigated using in vitro and in vivo assays. Structure-function analyses in Aim 3 will define how the domains within SAP mediate its participation in NKT cell signaling. These investigations will increase our understanding of NKT cell development and may enable the design of new treatments for XLP or other human diseases associated with quantitative or qualitative NKT cell defects, including immunodeficiency, autoimmunity and cancer.